OB-Fold Used As Scaffold For Engineering New Specific Binders

ABSTRACT

The present invention pertains to the field of protein engineering, and provides means for obtaining stable molecules that specifically bind to a target selected amongst a large variety of ligands families. In particular, the present invention provides methods for obtaining a molecule specifically binding to a target of interest, through a combinatorial mutation/selection approach with an OB-fold protein as a starting molecule. In particular, the target of interest can be of a different chemical nature form that of the native target of the OB-fold protein used as the starting molecule.

RELATED APPLICATIONS

This application is divisional application of U.S. patent applicationSer. No. 12/517,643, which is the national stage (under 35 U.S.C. §371)of PCT/IB2007/004388, filed Dec. 4, 2007, which claims benefit ofEuropean application EP 06291869.3, filed Dec. 4, 2006. The entirecontents of each of these applications are hereby incorporated byreference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 1535129 Sequence Listing. The size of the textfile is 47 KB, and the text file was created on Aug. 11, 2015.

The present invention pertains to the field of protein engineering. Morespecifically, the invention provides means for obtaining stablemolecules that specifically bind to a target selected amongst a largevariety of ligands families.

Most natural proteins are not adapted to therapeutic or evenbiotechnological uses. The challenge of protein engineering is to defineefficient and comprehensive ways for the design of new or improvedproteins with the required properties. Among targeted properties,recognition of a ligand with high specificity and affinity is of premiumimportance. For many applications, antibodies have been used because oftheir extraordinary adaptability to binding very different kinds ofligands, such as proteins, peptides, nucleic acids, sugars, etc.However, antibodies or their derivative fragments, are often difficultand expensive to produce because of their molecular complexity and/ortheir insufficient stability. For these reasons, alternatives toantibodies have recently been developed using scaffold proteinsengineered via a combinatorial mutation/selection approach. The aim wasto maintain favorable properties of antibodies, while getting rid oftheir disadvantages. Successful applications of this strategy have beenreported (Binz et al., 2005; Mathonet and Fastrez, 2004), the vastmajority of targets being proteins and, exceptionally, small organiccompounds.

The present invention aims at providing tools for engineering moleculeswell adapted to medical or biotechnological uses, and able to bindspecifically and with a high affinity to various targets, selectedamongst nucleic acids, proteins, carbohydrates or any other biologicalmolecule. To this aim, the inventors lave tested the hypothesis that aparticular protein structure, named oligonucleotide/oligosaccharidebinding-fold (OB-fold), might be modified by randomization of theresidues of its binding face, while at least partially maintaining thefavorable biophysical properties of the parent protein (i.e., highstability and folding efficiency).

The oligonucleotide/oligosaccharide binding-fold (OB-fold), firstdescribed by Murzin (Murzin, 1993), is found in all kingdoms and wasranked as the 28th most represented fold in a survey of 20 genomes (Qianet al., 2001). This fold, a five stranded β-barrel capped with anamphiphilic α-helix, appears suitable for a wide diversity of sequences.The OB-fold presents a β-sheet binding face that confers remarkablediversity in the types of compounds that can be recognized: ssDNA,dsDNA, RNA, oligo saccharides, proteins, metallic ions and catalyticsubstrates. A study of several OB-folds from different proteins showedthat the binding core is always located at the same position of thebinding face Arcus, 2002).

Sac7d has an OB-fold topology (FIG. 1 a) (Agback et al., 1998; Gao etal., 1998; Robinson et al., 1998; Su et al., 2000). It belongs to aclass of small chromosomal proteins from the hyperthermophilic archaeonSulfolobus acidocaldarius. Sac7d binds double strand DNA without anyparticular sequence' preference, while inducing a sharp kink in the DNA(Robinson et al., 1998). It is thought to play a role in DNA helixstabilization at the high growth temperature of Sulfolobusacidocaldarius (optimal at 85° C.). Sac7d is extremely stable. It isthermostable and unfolds with a T_(m) of 91° C., it maintains a nativefold between pH 0 and 10, and its guanidinium hydrochloride induceddenaturation occurs reversibly with a midpoint concentration of 2.8Mdenaturant (McCrary et al., 1996). Contrary to antibodies, its molecularorganization is quite simple. Sac7d is a small monomeric protein of 66amino acids, with one structural domain (i.e., the OB-fold), and doesnot have a disulfide bridge (McAfee et al., 1995). Several truncatedforms of Sac7d have been observed (McAfee et al., 1995). Overproductionlevels up to 10-15 mg of soluble protein per liter flask culture ofEsherichia coli can be reached (Edmondson and Shriver, 2001). Structuralstudies of Sac7d and its close homologue Sso7d from Sulfolobussolfataricus have shown that the two protein cores are superimposableand that binding to the minor groove of DNA occurs mainly via a twistedarea formed by 16 residues (Agback et al., 1998; Gao et al., 1998;Robinson et al., 1998).

The inventors have explored the possibility to change the bindingspecificity of Sac7d by in vitro directed protein evolution, through theintroduction of random mutations in a number of residues involved in theligand binding, followed by a selection of the variants which bind to agiven target. More precisely, the inventors have constructed a largelibrary of about 3.10¹² variants corresponding to random substitutionsof 11 residues of the binding interface of Sac7d. This library has beenused to select variants able to bind a defined protein target, byribosome display. Pools of binders for three protein targets wereobtained, with affinities in the hundred nanomolars range. In a secondapproach, the inventors extended the potential binding area up to 13 and14 residues. These new libraries were used for selection on one of theprevious targets (PulD-N), and specific binders with affinity in thepicomolar range were obtained. Hence, the inventors have demonstratedthat, surprisingly, a general DNA binder (Sac7d) can be evolved towardsa specific protein binder having high specificity and affinity. Thisdemonstration provides a proof of principle that binders for virtuallyany kind of ligand can be derived from Sac7d or other OB-fold proteins,for example by using the process described in more detail below.

A first aspect of the present invention is hence the use of an OB-foldprotein, as a starting molecule for obtaining, through a combinatorialmutation/selection approach, a molecule specifically binding to atarget, especially to a target to which the starting OB-fold proteindoes not bind, i.e., to a target different form the target of theOB-fold protein used as the starting molecule. In what follows, thetarget of the starting OB-fold protein will be called the “nativetarget”, whereas the target used in the selection step will bedesignated as the “target of interest”. In a preferred embodiment, thetarget of interest is of a chemical nature different from that of thenative target (for examples, protein vs./nucleic acid, and metallic ionor sugar vs./protein). A particular aspect of the present invention ishence the use of an OB-fold protein naturally binding to a nucleic acid,as a starting molecule for obtaining, through a combinatorialmutation/selection approach, a molecule specifically binding to a targetdifferent from a nucleic acid (for example, a protein). The use of anOB-fold protein naturally binding to a protein, as a starting moleculefor obtaining, through a combinatorial mutation/selection approach, amolecule specifically binding to a non-protein target (for example, anucleic acid) is also part of the present invention.

According to the present invention, the phrase “OB-fold protein”designates any polypeptide which comprises or consists of a domainhaving OB-fold topology, as described by (Murzin, 1993) and (Arcus,2002). This topology corresponds to an architecture which comprises afive-stranded (β-barrel capped at one end by an amphiphilic α-helix.Referring to the CATH protein structure classification (Pearl et al.,2003), the OB-fold topology corresponds to the 2.40.50 fold family (CATHdatabase version 3.0.0: Released May 2006). Such an OB-fold protein canbe either a native protein (i.e., an isolated, purified or recombinantprotein having the same sequence as a natural protein), or an engineeredprotein (like, for example, a fragment of a native protein, or a fusionprotein comprising an OB-fold domain from a first protein, and anothermoiety from another protein). Non-limitative examples of OB-foldproteins which can be used according to the invention are Sac7d, Sso7d,the N-terminal domain of SEB (Papageorgiou et al., 1998), the chain A ofthe Shiga-like toxin IIe (PDB 2bosa), the human Neutrophil ActivatinPeptide-2 (NAP-2, PDB 1tvxA), the Molybdenum Binding Protein (modg) ofAzotobacter vinelandii (PDB 1h9j), the N-terminal domain of SPE-C(Roussel et al., 1997), the B₅ subunit of E coli Shiga-like toxin (Kitovet al., 2000), Cdcl3 (Mitton-Fry et al., 2002), the cold-shockDNA-binding domain of the human Y-box protein YB-1 (Kloks et al., 2002),the E coli inorganic pyrophosphatase EPPase (Samygina et al., 2001), orany of the proteins listed in Table 3 of the article by (Arcus, 2002),such as 1krs (Lysyl-tRNA synthetase LysS, E. coli), 1c0aA (Asp-tRNAsynthetase, E. coli), 1b8aA (Asp-tRNA synthetase, P. kodakaraensis),llylA (Lysyl-tRNA synthetase LysU, E. coli), 1quqA (Replication proteinA, 32 kDa subunit, Human), 1quqB (Replication protein A, 14 kDa subunit,Human), 1jmcA (Replication protein A, 70 kDa subunit (RPA70) fragment,Human), lotc (Telomere-end-binding protein, O. nova), 3ullA(Mitochondrial ssDNA-binding protein, Human), 1prtF (Pertussis toxin S5subunit, B. pertussis), 1bcpD (Pertussis toxin S5 subunit (ATP bound),B. pertussis), 3chbD (Cholera Toxin, V. cholerae), 1tiiD (Heat-labiletoxin, E. coli), 2bosA (Verotoxin-1/Shiga toxin, B-pentamer, E. coli), 1br9 (TIMP-2, Human), lan8 (Superantigen SPE-C, S. pyogenes), 3seb(Superantigen SPE, S. aureus), law7A (Toxic shock syndrome toxin, S.aureus), 1jmc (Major cold-shock protein, E. coli), 1bkb (Initiationtranslation factor 5a, P. aerophylum), 1 sro (Si RNA-binding domain ofPNPase, E. coli), 1d7qA (Initiation translation factor I, elF1a, Human),1ah9 (Initiation translation factor I, IF1, E. coli), 1b9mA(Mo-dependent transcriptional regulator ModE, E. coli), 1ckmA (RNAguanylyltransferase, Chlorella virus, PBCV-1), 1a0i (ATP-dependent DNAligase, Bacteriophage T7), 1snc (Staphylococcal nuclease, S. aureus),1hjp (DNA helicase RuvA subunit, N-terminal domain, E. coli), 1pfsA(Gene V protein, Pseudomonas bacteriophage pf3), 1gvp (Gene V protein,Filamentous bacteriophage (fl, M13)), 1gpc (Gene 32 protein (gp32) core,Bacteriophage T4), 1wgjA (Inorganic pyrophosphatase, S. cerevisiae), and2prd (Inorganic pyrophosphatase, T. thermophilus). It can be noted thatOB-folds domains originating from toxins can be used as startingmolecules even for purposes in which toxicity is to be avoided, sincemutations in their binding site, and hence change in their bindingspecificity, can completely abolish their toxicity.

As exemplified in more detail in the experimental part below, thecombinatorial mutation/selection approach consists in obtaining acombinatorial library corresponding to the randomization of a number ofchosen residues of the starting OB-fold protein (especially,randomization of a number of residues involved in the binding of theprotein with its native ligand), followed by a selection, in saidlibrary, of variants which have the desired properties.

Another object of the present invention is hence a combinatorial librarycorresponding to the randomization of 5 to 32, preferably 8 to 20, andmore preferably 11 to 16 residues of the binding interface of an OB-foldprotein with its native ligand, possibly combined with the deletion of 1to 4 residues and/or the insertion of 1 to 50 residues. Of course, the“binding interface of an OB-fold protein” herein designates, even incases of proteins with multiple domains binding to different ligands,the interface between the OB-fold domain and the ligand which binds tothis domain. The skilled artisan will find in the scientific literaturethe necessary information to identify the residues involved in thebinding of the OB-fold protein with its native ligand, which are oftenlocated in β-strands β3, β4 and β5 and in loops 1, 3 and 4 of theOB-fold (FIGS. 1 b and 2).

Particular combinatorial libraries according to the present inventioncan be obtained with Sac7d from Sulfolobus acidocaldarius, or with itshomologues (FIG. 4) such as Sso7d Sso7d from Sulfolobus solfataricus. Ofcourse, libraries obtained with a truncated form of Sac7d, such as thosedescribed by (McAfee et al., 1995), are also part of the presentinvention.

By superimposing several sequences and 3D-structures of OB-fold domainsusing the web sites WU-Blast2 (http://www.ebi.ac.uk/blast2/index.html)(Lopez et al., 2003), T-COFFEE(http://www.ch.embnet.org/software/TCoffee.html) (Notredame et al.,2000) and DALI lite (http://www.ebi.ac.uk/DaliLite/) (Holm and Park,2000), the inventors have identified the positions which could bemodified for obtaining the libraries according to the present invention.Taking as a reference the sequence of Sac7d of SEQ ID No: 1, theresidues which can be randomized are the following: V2, 1K3, K5, K7, Y8,10, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36,N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50 and P51.

Still with Sac7d as a reference, the residues which can be deleted are:A59, R60, A61 and E64.

A superimposition of 3D structures of 10 proteins or OB-fold domains(including Sac7d), using the web site DALI(http://wvvw.ebi.ac.uk/dali/Interactive.html) (Holm and Sander, 1998),revealed that this kind of proteins have loops of various sizes, whichmost probably are involved in the binding of ligands (see FIG. 2). Thisobservation is consistent with previously published data (Arcus, 2002).Since Sac7d is one of the proteins having the shortest loops, randominsertions in said loops can advantageously be performed in order toobtain libraries particularly adapted for the binding of certain ligandsfamilies. Advantageously, insertions of 1 to 15 amino acid residues canbe performed in loop 3 (as identified in FIGS. 1 b and 2), for examplein the region of residues 25 to 30 of Sac7d, preferably between residues27 and 28, insertions of 1 to 15 amino acid residues can be performed inloop 4 (as identified in FIGS. 1 b and 2), for example in the region ofresidues 35 to 40 of Sac7d, preferably between residues 37 and 38, andinsertions of 1 to 20 amino acid residues can be performed in loop 1 (asidentified in FIGS. 1 b and 2), for example in the region of residues 7to 12 of Sac7d, preferably between residues 9 and 10. In order to avoidany ambiguity, it is herein specified that loops 3, 4 and 1 asidentified in FIGS. 1 a and 2 respectively correspond to loops 1, 2 and4 identified in the review article by Arcus (supra).

According to a particular embodiment of the invention, the combinatoriallibrary corresponds to the randomization of 11, 12, 13, 14, 15, 16, 17or 18 residues of Sac7d selected amongst K7, Y8, K9, K21, K22, W24, V26,K28, M29, S31, T33, K39, T40, R42,

A44, S46, E47 and K48, for example 11, 12, 13, 14, 15 or 16 residuesselected amongst K7, Y8, K9, K21, K22, W24, V26, K28, M29, S31, T33,K39, T40, R42, A44 and S46.

In a preferred combinatorial library of the above embodiment, therandomized residues comprise at least the residues K7, Y8, K9, W24, V26,M29, S31, T33, R42, A44 and S46 of Sac7d. A specific library accordingto this embodiment, and designated in the experimental part below as“library 11”, corresponds to the randomization of the residues K7, Y8,K9, W24, V26, M29, S31, T33, R42, A44 and S46 of Sac7d. In othercombinatorial libraries according to this embodiment, one, two or threeadditional residues of Sac7d selected amongst K21, 1(22 and T40 are alsorandomized. Two other preferred libraries according to the invention,also described in the experimental part, are “library 13”, whichcorresponds to the randomization of the residues K7, Y8, K9, K21, K22,W24, V26, M29, S31, T33, R42, A44 and S46 of Sac7d, and “library 14”,which corresponds to the randomization of the residues K7, Y8, K9, 1(21,K22, W24, V26, M29, S31, T33, T40, R42, A44 and S46 of Sac7d.

Of course, a combinatorial library obtained by randomizing 11, 12, 13,14, 15 or 16 residues of Sso7d selected amongst the residues located atpositions which are equivalent to those listed above, is also part ofthe present invention. Said equivalent positions can be identified byusing the file for Sso7d (chain A) in the RSCB Protein Data Bank (Bermanet al., 2000) (1 bf4A), and by comparing its 3D-structure with that ofSac7d (FIG. 3 a):

Sac7d: 7 8 9 21 22 24 26 28 29 31 33 39 40 42 44 46 Ibf4A: 7 8 9 21 2224 26 28 29 31 33 40 41 43 45 47

Other proteins homologous to Sac7d can also be used for obtainingcombinatorial libraries as described above. Examples of such proteinsare disclosed in the following Table.

TABLE 1 Examples of Sac7d homologues which can be used according to thepresent invention Primary Secondary Accession Accession Name Sourcenumber Number Sac7d: (DNA- Sulfolobus P13123 Q4JCI7 binding protein 7d)acidocaldarius NCBI: AAA80315 Sac7e: (DNA- Sulfolobus P13125: Q4JBQ1binding protein 7e) acidocaldarius NCBI: YP_255071 DNA-binding_protein_7Sulfolobus Q96X56 (DBP 7) tokodaii NCBI: Q96X56 Ssh7b (DNA- SulfolobusO59632 binding protein 7b) shibatae NCBI: BAA28275 Sso7d (DNA-Sulfolobus P39476 P81550 binding protein 7d) solfataricus NCBI: P39476Ssh7a (DNA- Sulfolobus P61990 O59631, binding protein 7a) shibataeP80170 NCBI: BAA28274 Q9UWI8 p7ss (DNA- Sulfolobus P61991 O59631,binding protein 7a) solfataricus P80170, NCBI: P61991 Q9UWI8

Alignment of the OB-fold domains of the Sac7d homologues listed in Table1 is shown in FIG. 4.

Other particular combinatorial libraries according to the presentinvention can be obtained with the Shiga-like toxin IIe (PDB 1r4pB), forexample by randomizing 11, 12, 13, 14, 15 or 16 residues selectedamongst the residues located at positions which are equivalent to thoselisted above for Sac7d, said equivalent positions (FIG. 3 b) being asfollows:

Sac7d: 7 8 9 21 22 24 26 28 29 31 33 39 40 42 44 46 lr4pB: 60 59 58 9 1012 14 17 18 20 22 X X 27 29 31

Other examples of particular combinatorial libraries according to thepresent invention can be obtained by randomizing 11, 12, 13, 14, 15 or16 residues of the human Neutrophil Activating Peptide-2 (NAP-2, PDB1tvxA), wherein said residues are selected amongst the residues locatedat positions which are equivalent to those listed above for Sac7d. Theseequivalent positions, obtained by comparison of the 3D structure of saidprotein with that of Sac7d (FIG. 3 c), are the following:

Sac7d: 7 8 9 21 22 24 26 28 29 31 33 39 40 42 44 46 ltvxA: 25 26 27 4041 43 45 54 55 57 59 61 63 65 67 69

Still other particular combinatorial libraries according to the presentinvention can be obtained with the Molybdenum Binding Protein (modg) ofAzotobacter vinelandii (PDB 1h9j), for example by randomizing 11, 12,13, 14, 15 or 16 residues selected amongst the residues located atpositions which are equivalent to those listed above for Sac7d, whereinsaid equivalent positions (FIG. 3 d) are as follows:

Sac7d: 7 8 9 21 22 24 26 28 29 31 33 39 40 42 44 46 1h9jA: 60 61 62 8687 89 91 93 94 96 98 105 106 108 110 112

The present invention also pertains to the use of a combinationallibrary as those described above, for engineering a moleculespecifically binding to a target. In the present text, a molecule isconsidered as a “specific” binder of a target, if it binds to it with asignal to noise ratio superior to 10. A binder molecule which isengineered by using a combinatorial library according to the inventionpreferably binds to its target (also called “target of interest”) with ahigh affinity, i.e., with an affinity better than 10 nM when the targetis a peptide or a protein, and better than 1 μM when the target is acarbohydrate molecule, for example.

Another object of the invention is a process for engineering a moleculespecifically binding to a target, comprising a step of selecting, in acombinational library as described above, those variants of said OB-foldprotein which specifically bind to said target. By “variant” is hereinmeant a protein belonging to the library, i.e., a protein which isderived from the starting OB-fold protein by mutations in its bindingsite.

As already mentioned above, one of the interests of the presentinvention is that it enables the engineering of a molecule whichspecifically binds to a target of interest, with virtually no limitationconcerning this target. For example, the target of interest can be apeptide, a protein, an oligosaccharide, a lipid, a lipopeptide, acarbohydrate (for example, a sugar), a single-stranded DNA, adouble-stranded DNA, a RNA. Interestingly, the target of interest can bea natural or synthetic small molecule, limited to a few atoms, or evento only one atom. Examples of such small molecules include any kind ofhaptens (i.e., small molecules which can elicit an immune response onlywhen attached to a large carrier), vitamins, or metallic ions. The widediversity of molecules that can be used as targets is especiallyinteresting, since the OB-fold proteins binding to these targets can beused either for the same applications as already known for antibodies,or for different applications. For example, OB-fold proteins obtainedaccording to the present invention and binding to metallic ions can beused in bioremediation processes, for example to remove heavy metalsfrom a complex material such as soil or polluted water.

The skilled artisan can use any technique of the art to perform theselection step of the process according to the invention and/or toperform the screening step for obtaining a protein with a desiredproperty. Selection techniques can be, for example, phage display(Smith, 1985), mRNA display (Wilson et al., 2001), bacterial display(Georgiou et al., 1997), yeast display (Boder and Wittrup, 1997) orribosome display (Hanes and Pluckthun, 1997).

Ribosome display is particularly advantageous for performing theselection step, since it is performed completely in vitro and thuscircumvents many limitations of in vivo systems (especially regardingthe library size) (He and Taussig, 2002; Schaffitzel et al., 1999). Theskilled artisan will find in the experimental part below, as well as inthe articles by He and Taussig (2002), and Schaffitzel et al. (1999), orin other articles and manuals, protocols for performing ribosomedisplay.

In a preferred embodiment of the process of the invention, 2, 3, 4 or 5rounds of selection are performed by ribosome display.

As described in the experimental part, the process according to theinvention can also comprise a further step of isolating andcharacterizing one or several molecules specifically binding to thetarget of interest. For example, the isolation and characterizationsteps can be performed as follows:

(i) mRNAs from a pool selected by ribosome display are reversedtranscribed and amplified by PCR, and the resulting DNAs are cloned intoexpression vectors;

(ii) bacteria (for example, competent DH5α) are transformed by saidexpression vectors (comprising said DNAs) and individual clones aregrown; and

(iii) proteins extracted from said bacterial clones are tested for theirbinding to the target and/or other biological properties (such asstability and the like).

The clones expressing the proteins which have the best properties canthen be grown to produce larger amounts of proteins. The coding sequenceof the engineered protein can be determined by sequencing the pertinentpart of the expression vector, and the gene encoding this protein canalso be used for further engineering of the protein. Indeed, it can beadvantageous to use a binder obtained through a process according to theinvention to construct multifunctional proteins having at least onetargeting moiety and another moiety with catalytic, fluorescent,enzymatic or any other kind of activity. This can be made, for example,through the construction of a fusion protein comprising, as a firstmoiety, the isolated and characterized molecule, and at least a secondmoiety. This second moiety can be advantageously selected amongstenzymes, markers or reporter proteins (such as PhoA, GFP and the like),therapeutic proteins, etc. According to a variant of the process of theinvention, several binders are linked together (either in a fusionprotein, or through non-covalent links), in order to construct fusionsor complexes with multiple binding specificities.

Other objects of the invention are proteins obtained through a processas described above. In particular, proteins which specifically bind toPulD, and which have a sequence selected amongst SEQ ID Nos: 2 to 8, arepart of the present invention, as well as the GarA-binder of SEQ ID No:47.

The invention is further illustrated by the following figures andexamples.

FIGURE LEGENDS

FIG. 1: a) Schematic representation of wild type Sac7d in complex withdouble strain DNA (PDB code 1azp). b) Residues participating to DNAbinding. The concave area is defined with residues coloured in lightgrey, the flat area with those coloured in medium and dark grey. Theresidues at limit between the regions of the binding area are colouredin medium grey.

FIG. 2: Schematic representation of loops 1, 3 and 4 of various OB-folddomains. The RSCB PBD structure files used for performing thissuperimposition are the following:_(—)1azp (i.e., Sac7d), 1tvxA, 1dokA,1bqq, 1csp, 3seb, 1br9, 2bosA, 1bf4A and 1quqB. For clarity of thefigure, only the structure of Sac7d is represented.

FIG. 3: Schematic representation and superimposition with Sac7d of a)Sso7d (structure file: 1bf4); b) Shiga-like toxin He (structure file:2bosa); c) human Neutrophil Activating Peptide-2 (NAP-2, structure file:1tvxA); d) Molybdenum Binding Protein (modg) of Azotobacter vinelandii(structure file: 1h9j). Nota bene: for proteins which compriseadditional domains different from the OB-fold domain, only the OB-folddomain is shown in the figure. The structure superimpositions wereobtained using DALI and DALI Lite servers. The protein sequences, withtheir numbering in the NCBI file, are shown in Table 2 below.

TABLE 2 Protein sequences which have been used for obtaining thesuperimpositions shown in FIG. 3. The sequence fragments in italicscorrespond to the sequences effectively used in the structure fileused to determine the positions which could advantageously berandomly mutated. Said mutation positions are in bold. Name File ref.Source Sequence Sac7d 1azp NCBI: MVKVKF

GEEKEVDTSKI

V

R

G

V

AAA80315 F

YDDNG

G

G

V

EKDAPKELLDMLARAE REKK (SEQ ID No: 1) Sso7d 1bf4 NCBI: MATVKF

GEEKEVDISKI

V

R

GKMI

P39476 F

YDEGGG

G

G

V

EKDAPKELLQMLEKQ KK (SEQ ID No: 28) Toxin 1r4pb NCBI:MKKMFMAVLFALVSVNAMAADCAKGKI

S

IIe CAA90631 Y

ED

F

V

VDGK

Y

T

RWNLQPLLQSAQ LTGMTVTIKSSTCE

GFAEVQFNND (SEQ ID No: 29) NAP-2 1tvxA NCBI:MSLRLDTTPSCNSARPLHALQVLLLLSLLLT CAG33086 ALASSTKGQTKRNLAKGKEESLDSDLYAELR

IKTTSGIHPKNI

L

V

GKGTHCNQ

V

A

L

D

R

I

L

PDAPRIKKIVQKKLAGD ESAD (SEQ ID No: 30) modg 1h9j NCBI:MKISARNVFKGTVSALKEGAVNAEVDILLGG CAA90038 GDKLAAVVTLESARSLQLAAGKEVVAVV

WVLLMTDSSGYRLSARNILTGTV

I

T

A

A

V

LALQGG

I

S

V

KEAVAELGLKPG ASASAVIKASNVILGVPA (SEQ ID No: 31)

FIG. 4: Alignment of the OB-fold domains of several Sac7d homologues.FIG. 4 discloses SEQ ID NOS: 33-38, respectively, in order ofappearance.

FIG. 5: Gene synthesis of the mutated sequences encoding Sac7d.Positions randomized for the library 11 are labelled in black.Additional randomized positions for library 14 are labelled in grey.Oligonucleotides used are represented with thin arrows (see text fortheir sequences). The large arrow corresponds to the coding sequence ofSac7d (whitout the C-ter TolA linker).

FIG. 6: a) RIA (radioimmuno assay) after four rounds of selection withlibrary 11 against DnaK, PulD-N and GarA. Pools of selections weretranslated in vitro in presence of methionine ³⁵S and were tested forbinding to DnaK, PulD-N or GarA immobilized in an ELISA plate. Afterwashing and elution with 0.1 M triethanolamine, amounts of binders wereestimated with a β counter. Competitions were carried out in parallelwith pre-incubation of translated pools with free proteins atconcentrations of 1 μM and 10 μM. b) Screening of anti-PlulD-N bindersafter round 4. c) Test of binding specificities for six anti PulD-Nbinders by ELISA. Interaction between binders and immobilized DnaK,PulD-N, GarA and BSA were compared.

FIG. 7: RIA after five rounds of selection with libraries 11, 13 and 14against PulD-N. Pools of selections were translated in vitro in presenceof methionine ³⁵S and were tested for binding to DnaK, PulD-N, GarA orBSA immobilized in an ELISA plate. After washing and elution 0.1 Mtriethanolamine, amounts of binders were estimated with a 13 counter.Competitions were carried out in parallel with pre-incubation oftranslated pools with free PulD-N at 1 nM, 10 nM, 100 nM, 1000 nM. BSAwas used as negative control for binding of pools (no competition done).

FIG. 8: Sequences of eight selected binders against PulD-N. The sequenceof the wild type Sac7d is shown at the top of the figure. Residuescommon to the wild type Sac7d and binders are represented by a dot.Positions that were programmed in the substitution scheme arehighlighted in grey. FIG. 8 discloses SEQ ID NOS: 33 and 39-47,respectively.

FIG. 9: Expression and purification analysis of eight selected binders.Proteins were loaded on a 15% SDS-PAGE stained with coomasie brilliantblue. a) E. coli crude extract after expression at 30° C. Cells wereharvested after 19h induction with 0.5 mM IPTG and lyzed in loadingbuffer. b) Soluble fractions of crude extracts prior to purification ofbinders. c) Purified binders after one step IMAC (immobilized metal ionaffinity chromatography) purification of the E. coli crude extractsoluble fraction. All loaded samples were equivalent to 13 μl liquidculture.

FIG. 10: Affinity determination of clone 6 using SPR (surface plasmonresonance) analysis. a) The kinetics of binding were followed using aBIAcore. Biotinylated PulD-N was immobilized (250 RU) on a streptavidinchip and clone 6 was injected at 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nMand 1.56 nM. A flow cell without immobilized PulD-N was used to controlthat no specific binding occurred and the signal from cell wassubtracted to the one obtained from the measurement cell. BIAevaluationsoftware was used to analyze data with a global fitting procedure. b)The affinity was also measured at equilibrium using competition BIAcore.The determined parameters from both approaches are reported in Table 3.

FIG. 11: Thermal stabilities of wild-type and variants Sac7d recombinantproteins determined by differential scanning calorimetry. The excessheat absorption of wt Sac7d, clone 40, clone 33, and clone 6, arereported at 1° C./min at a protein concentration of 200 pg/ml in 50 mMMES buffer pH 5.6 with 300 mM NaCl. For each protein sample, theexperimental excess heat capacity curve (thick line) was best fitted toa non two-state model (thin line) by non-linear regression. Resultingthermodynamic parameters are given in Table 4.

FIG. 12: a) Western blot for detection of PulD-N mixed with E. colicrude extract using clone 6-alkalin phosphatase fusion. b) Single steppurification of PulD-N with clone 6 immobilized on a column. The solublefraction of an E. coli extract containing PulD-N was injected on thecolumn equilibrated with HBS buffer pH7.0. After washing, the pH wasdropped to 2.5 with a glycine-HCl buffer. Fractions were analyzed on a15% SDS-PAGE and stained with coomasie brilliant blue. CE: cell extract;PulD-N: pure protein used as marker; Fractions: eluted fractions fromthe column after acidic pH jump; FT: flow through.

FIG. 13: Binding of Sac7*40, Sac7*33, and Sac7*6 to isolated cellenvelopes and to PulD dodecamers (PulDD) and PulD monomers (PulDM). (A)Increasing amounts of cell envelope from strain PAP105 producing PulDand PulS or PulD-CS and PulS were incubated with Sac7*40-GFP, and themembrane fraction was then analyzed by SDS and immunoblotting with GFPantibodies. (B) Far-Western blotting of the indicated amounts of cellenvelope proteins from the same strains using the three Sac7*-PhoAchimeras and antibodies against PhoA

FIG. 14: Production of Sac7-PhoA chimeras with and without IPTGinduction and their effects on secretion and PulD multimerization incell envelope protease deficient strain PAP5198 carrying pCHAP231. (A)Sac7-PhoA levels detected by immunoblotting (with PhoA antibodies) ofthe same amount of cell extract. (B) Levels of secretion (%) andpresence of PulD dodecamers (PulDD) and monomers (PulDM) detected byimmunoblotting of phenol-treated and nontreated cell extracts with PulDantibodies. Arrows indicate PulDM detected without phenol treatment. Sindicates Sac7d-PhoA. (C) As B, but without IPTG induction.

FIG. 15: PCR carried out on ADNc obtained from ARNms, eluted duringcycle No.4 (see FIG. 19, RT-PCR stage).

FIG. 16: competitive radio-immune tests carried out after five anti-PKnGand anti-lysozyme selection cycles.

FIG. 17: ELISA screening of anti-lysozyme (A), anti-PknG (B) andantiGarA (C) clones obtained after 5 selection cycles

FIG. 18: anti-lysozyme clone sequences obtained after five selectioncycles. FIG. 18 discloses SEQ ID NOS: 49-61, respectively, in order ofappearance.

FIG. 19: Alignment of a GarA-binder with Sac7d. FIG. 19 discloses SEQ IDNOS: 62, 47 and 63, respectively, in order of appearance.

FIG. 20: Competition ELISA after the 4^(th) round of selection againstFc fragment.

FIG. 21: Schematic representation of a round of ribosome displayselection.

EXAMPLES

The experimental data which follow have been obtained using the materialand methods described below.

Materials and Methods

General Molecular Biology

Enzymes and buffers were from New England Biolabs (USA) or Fermentas(Lithuania). Oligonucleotides were from MWG Biotech (Germany). All PCRs(polymerase chain reactions) were performed using Vent polymerase if notindicated in the text. The cloning and expression vector for thewild-type Sac7d and selected mutants was pQE30 from Qiagen (Germany).

Synthesis of the DNA Encoding the Wild Type Sac7d

DNA Sequence of the wild type Sac7d was generated by assembly PCR usingthe six following oligonucleotides:

SC1 (SEQ ID No: 9: GAAACTCCTAGGTATTGTGCTGACGACCCCGATCGCGATCTCTAGCTTTGCGGTGAAAGTGAAA TT),SC2 (SEQ ID No: 10: GATCTTGCTGGTGTCCACTTCTTTTTCTTCGCCTTTATATTTAAATTTCACTTTCACCGCAAAGCT AG),SC3 (SEQ ID No: 11: GAAGTGGACACCAGCAAGATCAAGAAAGTTTGGCGTGTGGGCAAAATGGTGAGCTTTACCTACGACGACAACGGCAAG),SC4 (SEQ ID No: 12: CTCTTTCGGGGCATCTTTCTCGCTCACGGCGCCACGGCCGGTCTTGCCGTTGTCGTCGTA),SC5 (SEQ ID No: 13: GAGAAAGATGCCCCGAAAGAGTTATTAGATATGTTAGCGCGTGCGGAAAGCTTCAACCA),SC6 (SEQ ID No: 14: TGGTGGTTGAAGCTTTCCGCACG).The purified PCR product served as template for a second PCRamplification using the two following primers:SC07 (SEQ ID No: 15: ATTAATGGTACCGGATCCGTGAAAGTGAAATTTAAA TATAAAG) andSC08 (SEQ ID No: 16: ATAATTGAGCTCTAAGCTTTTTTTCACGTTCCGCACGCGCTAACATATC). The PCR product was cloned into pQe30 expressionvector using BamHI and HindIII restriction sites. A clone with theexpected sequence was used for subsequent expressions.

Generation of Combinatorial Libraries

The protocol was the same for the generation of the three libraries 11,13, 14 in a format compatible with ribosome display. Libraries weremainly constructed by gene synthesis and PCR assembly steps. With asingle step PCR using a combination of four standards and threedegenerated oligonucleotides encoding NNS triplets (wherein N=A,C,T orG, and S=C or G), a DNA product including the 5′-flanking regionnecessary for ribosome display and the randomized gene of Sac7d wasobtained. For the library 14, the following oligonucleotides were used:

T7C (SEQ ID No: 17: ATACGAAATT AA TACGACTCACTATAGGGAGACCACAACGGTTTCCCTC),SDA_MRGS (SEQ ID No: 18: AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATATCCATGAGAGGATCG),SClib1 (SEQ ID No: 19: GGAGATATATCCATGAGAGGATCGCATCACCATCACCATCACGGATCCGTCAAGGTGAAA TTC),SClib2 (SEQ ID No: 20: GGATCCGTCAAGGTGAAATTCNNSNNSNNSGGCGAAGAAAAAGAAGTGGACACTAGTAAGATC),SClib3 (SEQ ID No: 21: CTTGCCGTTGTCGTCGTASNNAAASNNCACSNNTTTGCCSNNACGSNNAACSNNSNNGATCTTACTAGTGTCCACTT C),SClib4 (SEQ ID No: 22: TAATAACTCTTTCGGGGCATCTTTCTCSNNCACSNNGCCSNNGCCSNNCTTGCCGTTGTCGTCGTA),SClib5 (SEQ ID No: 23: CCATATAAAGCTTTTTCTCGCGTTCCGCACGCGCTAACATATCTAATAACTCTTTCGGGGCATC).Primers encoding wild-type triplets instead of NNS triplets at positionscorresponding to residues 21, 22 and 40, or to residue 40 in Sac7d wereused for the construction of libraries 11 and 13, respectively.

To the aim that the protein displayed on ribosome be accessible topotential ligands, the protein needs to be fused to a linker. Thesequence of this linker, corresponding to a part of the E. coli proteinTolA, encoded in the plasmid pRDV vector (Binz et al., 2004a),wasPCR-amplified using primers SClink (SEQ ID No: 24:GCGGAACGCGAGAAAAAGCTTTATATGGCCTCGGGGGCC) and tolAk (SEQ ID No: 25:CCGCACACCAGTAAGGTGTGC GGTTTCAGTTGCCGCTTTCTTTCT) (the latter encoding the3′-flanking region necessary for ribosome display). Finally the librarywas assembled with tolA linker by PCR assembly using tolAk and T7B (SEQID No: 48: ATACGAAATTAATACGACTCACTATAGGGAGA CCACAACGG) primers. Thefinal assembly product corresponded to a library of Sac7d with all the5′- and 3′- regions necessary to its use for ribosome display selectionsas previously described (Hanes et al., 1998; Schaffitzel et al., 1999).

Ribosome Display Selection Rounds

For selection experiments, biotinylated target proteins were used. Thebiotinylation was performed by incubation of a 10 μM solution of DnaK,GarA or PulD-N with a 20-fold molar excess ofSulfosuccinimidyl-6-(biotinamido) hexanoate (Sulfo-NHS-LCLC-Biotin,Pierce) in PBS on ice for 1 h. The biotinylated proteins werebuffer-exchanged using protein desalting spin columns from Pierceequilibrated in TBS. The degree of biotinylation was determined usingthe HABA assay (Sigma) as from 2 to 3 molecules biotin per proteinmolecule. Biotinylated target proteins were bound to immobilizedneutravidin in a Maxisorp plate (Nunc) and selections by ribosomedisplay were performed at 4° C. essentially as described (Binz et al.,2004b) with some modifications. Briefly, after each round of selectionthe eluted mRNA was reverse-transcribed to cDNA and amplified using SDAMRGS and SClib5 primers. The PCR product was desalted on a NuclespinExtractII column (Macherey-Nagel) and used for a PCR assembly withTolAlinker DNA fragment (see above). This generated a full lengthribosome display construct with newly added 5′- and 3′-flanking regionsto minimize loss of clones due to degradation of the extremities of mRNAduring its manipulation. The number of RT-PCR cycles were 35 cycles(round 1), 30 cycles (round 2), 30 cycles (round 3), 25 cycles (round4). In round five, the selection pressure was increased using off-rateselection as described (Jermutus et al., 2001). For this selection, 10nM of biotinylated PulD-N was added to the stopped translation of thepool from round 4. The mix was equilibrated 1 h at 4° C. andnon-biotinylated PulD-N was added to a final concentration of 10 μM(1000 fold excess over biotinylated PulD-N). After 2 h incubation at 4°C. with agitation, the ternary complexes (mRNA-ribosome-binder) bound tobiotinylated PulD-N were capture with 30 μl of magnetic streptavidincoated beads (Roche) 15 min at 4° C. Beads were washed and mRNA wasisolated as for the first four rounds. The number of RT-PCR cycles was35 for this 5th round of selection. Progresses of selections werechecked by monitoring the amounts of RT-PCR products from round toround.

Analysis of Selected Pools and Isolated Clones

After four or five rounds, selected pools were tested by RIA(radioimmunoassay) as described (Hanes et al., 1998) using directlycoated target protein in a Maxisorp plate and using from 1 nM to 10 μMof free target protein as competitor.

The RT-PCR products from selected pools were cloned into pQE30 vectorusing BamHI and HindIII restriction sites and the resulting ligationswere used to transform E. coli DH5α strain. Clones were picked fromPetri dish to inoculate a deep-well plate containing 1.5 ml of LB mediumper well (100 μg/ml ampicillin, 1% glucose). After overnight culture at37° C. with shaking at 250 rpm, 0.2 ml of each well from this masterplate was used to inoculate an other deep-well plate containing 1.3 ml2YT medium (100 μg/ml ampicillin) per well. The plate was then incubatedat 37° C. for 1 with shaking (250 rpm). The expression was induced withthe addition of 0.5 mM IPTG and incubation at 30° C. for 4 h withshaking (250 rpm). Cells were pelleted with a centrifugation step(2250g) and supernatants discarded. Proteins were extracted with 50 μlBug Buster (Novagen) per well with shaking at 250 rpm for 30 min, then250 μl of TBS pH 7.4 (20 mM Tris-HCl, 150 mM NaCl) were added. Celldebris were pelleted with a centrifugation step (2500 g). For ELISA(enzymelinked immunosorbent assay) screening, 100 μl of each supernatantwere used to test the binding on target proteins coated into a Maxisorpplate. The detection was performed using the RGS His antibody HRPconjugate (Qiagen) that detects only the RGS-(His)₆-tag (SEQ ID NO: 64)from binders and not the (His)₆-tag (SEQ ID NO: 65) from target proteinsand BM-Blue substrate from Roche. All incubation steps were carried-outin TBS pH 7.4 with 0.1% Tween 20. Positive clones were sequenced bystandard sequencing techniques.

Protein Production for Screening by SPR (Surface Plasmon Resonance)

To perform the screening of binders based on their time of dissociationfrom PulD-N, clones were expressed in a 5 ml scale culture (E. coli DH5αstrain). Five hundreds microliters of an overnight preculture (LBmedium, 100 μl g/ml ampicilin, 1% glucose, 37° C.,) in deepwell wereused to inoculate 4.5 ml culture (2YT, 100 μg/ml ampicilin, 37° C.) indeepwell. Expression was induced at OD₆₀₀=1.0 by addition of 0.5 mM IPTGand cultures were incubated for 19 h at 30° C. (250 rpm). Cells wereharvested by centrifugation (2500 g) and proteins were extracted indeepwells by resuspension of cells in 0.5 ml TBS pH 7.4 containing 25 mMimmidazole, BugBuster and Benzonase (Novagen). After 1 h shaking at 4°C. the deepwell was centrifuged to pellet cell debris. Supernatants werepurified on microspin columns containing 100 μl Ni-Fast Flow ChelatingSepharose (General Electric) equilibrated with TBS pH 7.4 containing 20mM imidazole. Resin was washed 4 times with the loading buffer andpurified proteins were eluted with 400 μl TBS pH 7.4 and 250 mMimmidazole.

Protein Production and Purification of Binders and Wild-Type Sac7d

For Biacore and microcalorimetry experiments, the binders were expressedin DH5α E. coli strain in a 1 liter scale and purified as describedbelow. Fifty milliliters of an overnight pre culture (LB medium, 100μg/ml ampicilin, 1% glucose, 37° C.) were used to inoculate 1 literculture (2YT, 100 μg/ml ampicilin, 37° C.). Expression was induced atOD₆₀₀=1.0 by addition of 0.5 mM IPTG and cultures were incubated for 19h at 30° C. (250 rpm). Cells were harvested by centrifugation andresuspended in 30 ml TBS pH 7.4 at 30° C. containing 25 mM immidazole.Cells were lyzed with an Avestin Emulsiflex homogenizer and cell debriswere discarded with a centrifugation step. Proteins were purified on a 5ml HiTrap chelating column equilibrated with TBS pH 7.4 containing 25 mMimmidazole. Elution was performed with TBS pH 7.4 and 250 mM immidazole.Proteins were further purified by size-exclusion chromatography on aSuperdex 75 26/60 gel filtration column (GE Healthcare) equilibratedwith HBS pH 7.0 (20 mM HEPES, 150 mM NaCl). Analytical gel filtrationwere done on a SMART system (GE Healthcare) using a Superdex 75 3.2/20column equilibrated with HBS pH 7.0 (60 μl/min, 50 μl sample injected).BPTI (6.5 kDa), Ribonuclease A (14.6 kDa), Chymotrypsinogen A (20.3kDa), Ovalbumin (46.7 kDa) and Albumin (62.9 kDa) were used as molecularweight gel filtration calibration standards.

Surface Plasmon Resonance

SPR was measured using a BIAcore 2000 instrument at 25° C. BiotinylatedPulD was prepared as for selections by ribosome display and immobilizedon flow cells of a SA-chip. The densities of immobilized biotinylatedPulD-N for kinetic measurements, and for inhibition measurements were200 RU and 800 RU (saturated chip), respectively. The running buffer wasHBST pH 7.0 (20 mM HEPES, 150 mM NaCl, 0.05% Tween 20).

The screening and the ranking of binders according to their off-ratewere performed using micro IMAC purified proteins (see above) diluted1120 in running buffer prior injections for kinetics measurements at aflow rate of 60 μl/min.

Kinetic measurements for affinity determinations were performed withsize exclusion purified proteins injected at concentration ranging from1 nM to 50 nM. Inhibition measurements were done as described(Ostermeier et al., 1995) at a concentration of binder of 5 nM anddifferent concentrations of PulD-N as a competitor in concentrationranging from 35 0.2 nM to 20 nM at a flow rate of 25 μl/min. The slopeof the sensorgram for the binding phase was determined and plottedversus the concentration of PulD-N. Data evaluation was done usingBIAeval software (BIAcore).

Microcalorimetry

Differential scanning calorimetry was performed with a MicroCal VP-DSCcalorimeter as described previously (Hible et al., 2005). Stocksolutions of recombinant wildtype and variants Sac7d were dialyzedovernight at 4° C. against 50 mM MES buffer (PH 5.6 300 mM NaCl).Protein samples were then diluted to 200 μg/ml in the same bufferpreparation, degassed under vacuum for 10 min with gentle stirring priorto loading into the calorimeter cell (0.5 ml). The reference cell wasfilled with the same buffer. Samples were held in situ under a constantexternal pressure of 25 psi to avoid bubble formation and evaporation upto 130° C., equilibrated for 25 mM at 25° C., heated at a constantheating rate of 1 deg./min, and the data collected with a 16 sec filter.Data analysis was made with the Origin7™ software (Plotnikov et al.,1997) provided by the manufacturer. The excess heat capacity function(C_(p), excess) was obtained after subtraction of two base lines fromthe heat capacity function, the buffer reference thermogram and thecalculated chemical base line calculated after normalizing toconcentration from the progress of the unfolding transition.Thermodynamic parameters of the unfolding transitions of each proteinsample are the results of non-linear three-parameter (T_(m), ΔHcal,ΔH_(vH)) regression of the excess heat capacity curve assuming a nontwo-state model.

Construction of Alkaline Phosphatase Fusions

The genes encoding binders were PCR amplified with the primers SCPhoAF(SEQ ID No: 26: ATTAATGGTACCGGATCCGTGAAGGTGAAATTC) and SCPhoAR (SEQ IDNo: 27: ATAATTGAGCTCTAAGCTTTTTTTCACGCTCCGCAC) and Phusion polymerase tointroduce KpnI and SacI at 5′- and 3′-extremities, respectively. The PCRproducts were digested with KpnI and SacI and cloned into pQUANTagenvector (Qbiogene). Thus, alkaline phosphatase was fused to theC-terminus of the binder. Screening of alkaline phosphatase activeclones and periplasmic extractions were performed using DH5α E. colistrain and instructions from the manufacturer manual.

Briefly, positive clones were screened on LB plates (100 μg/mlampicillin, 4 μg/ml 5-bromo-4-chloro-3-indolylphosphate) for theiralkaline phosphate activity. Eight milliliters of an overnightpreculture (LB medium, 100 μg/ml ampicillin, 37° C.) was used toinoculate a culture of 400 ml (2YT medium, 100 μg/ml ampicillin, 1 gdipotassium phosphate pH 7.5, 37° C.). Induction of the tac promoter wasdone with addition of 0.5 mM IPTG when OD₆₀₀ was about 0.7 and thegrowth was continued for 4 h at 30° C. Cells were pelleted bycentrifugation and resuspended in 40 ml of TSE buffer (30 mM Tris-HCl pH8.0, sucrose 20%, 0.5 mM EDTA, 0.1 mg/ml lysozyme). The lysozymic shockwas performed by incubation of the suspension for 20 min at 4° C. withgentle agitation. Cell debris were discarded by centrifugation for 30min at 20000 g at 4° C. and supernatants were filtered through a 0.45 μmmembrane prior storage at −20° C.

Western Blots using Alkaline Phosphatase Fusions

A 10 ml overnight culture of DH5αE. coli strain (LB medium, 37° C.)without any expression vector was centrifuged to pellet cells. The cellpellet was resuspended in 1 ml TBS (1X BugBuster and 1 μl Benzonase).Cell lysis occurred at room temperature for 30 min and the suspensionwas centrifuged at 20000 g for 5 min to remove cell debris. Thesupernatant was then used for serial dilution of purified PulD-N.Samples were prepared with a constant volume of supernatant and avariable quantity of purified PulD-N corresponding from 0.4 ng to 400 ngPulD-N when 5 μI samples were loaded on a SDS gel. After migration,proteins were transferred from SDS gel to a nitrocellulose membrane(Hybond-C extra, 0.45 μm, General Electric). The membrane was blockedwith 5% dried milk in TBST (20 mM Tris-HCl, 150 mM NaCl, pH7.5, 0.1%Tween 20). The membrane was then incubated with soluble perimasplicextract of fusions diluted 1 to 15 in TBST milk for 1 h with gentleagitation. After washing the membrane, detection was done withprecipitating substrate NBT/BCIP (AP conjugate substrate kit, Biorad)diluted in reaction buffer (100 mM Tris-HCl pH9.5, 100 mM NaCl, 5 mMMgCl₂).

Construction and Expression of GFP (Green Fluorescent Protein) Fusions

The genes encoding binders were cloned via BamHI and HindIII sites intoa pQE30 derived plasmid (pFP3000) containing the gene for eGFP and aregion encoding for a flexible peptidic linker KLGSAGSAAGSGEF (SEQ IDNo: 32). This resulted in N-ter-binder-linker-eGFP-C-ter fusions with aMRGS (His)₆ tag (SEQ ID NO: 66) at the N-terminus. The sequences ofindividual clones were checked by DNA sequencing.

Expression and purification of binder-GFP fusions were done under thesame conditions as for binders alone (i.e., with an IMAC and a sizeexclusion chromatography step).

Isolation of Membrane Fractions and Interaction Studies with the 40-eGFPChimera

E. coli K-12 strains PAP105 (Guilvout et al., 1999) carrying theplasmids pCHAP3671 (pulD) and pCHAP580 (pulS) (Guilvout et al., 1999) orplasmids pCHAP3711(pulD-CS) (Guilvout et al., 2006) and pCHAP580 wereused for the isolation of the membrane vesicles. Cultures were grown toexponentially phase (OD₆₀₀: 0.9-1.1) in LB medium (Miller, 1992)containing appropriate antibiotics (100 μg/ml ampicillin, 25 μg/mlchloramphenicol) at 30° C. with vigorous aeration. The membranefractions were isolated by centrifugation (180,000×g for 30 min) afterFrench press desintegration of the cells and redissolved in Tris 50 mMpH 7.5, NaCl 150 mM at a final concentration of 450 μg/ml.

Different quantities of the membrane vesicles were incubated for 1 hourat room temperature with 70 μmol of the purified 40-eGFP fusion protein.After centrifugation (80,000×g for 20 min) the pellets were washed withthe same buffer, centrifuged again and resuspended in the same volume.The proteins in each sample were separated by SDS-PAGE and transferredonto nitrocellulose. The detection of the 40-eGFP fusion protein wasperformed by immuno-blotting with GFP primary antibodies.

Affinity Chromatography using Immobilized Clone 6 Binder

Seventeen milligrams of IMAC purified clone 6 was dialyzed against a 0.2M carbonate buffer pH 8.3 (0.5 M NaCl) and was injected on a 1 ml HiTrapNHS-activated HP column (General Electric) previously flushed with 6 mlof 1 mM HCl. Immobilization occurred at room temperature for 30 minutes.After washing the column and deactivating any remaining active groupswith 0.5 M ethanolamine (0.5 M NaCl, pH 8.3) and 0.1 M acetate (0.5 MNaCl, pH 8.3) for 30 min at room temperature, the column wasequilibrated with TBS pH 8.0 (20 mM Tris, 500 mM NaCl) and was ready touse for purification.

PulD-N protein was expressed using BL21(DE3) E. coli strain transformedwith the plasmid pCHAP3702 (Chami et al., 2005). Fifty milliliters of anovernight preculture (LB medium, 100 μg/ml ampicilin, 1% glucose, 37°C.) were used to inoculate 1 l culture (2YT, 100 μg/ml ampicilin, 37°C.). Expression was induced at OD₆₀₀=1.0 by addition of 1.0 mM IPTG andcultures were incubated for 4 h at 30° C. (250 rpm). Cells wereharvested by centrifugation and resuspended in 30 ml TBS pH 8.0. Cellswere lyzed with an Avestin Emulsiflex homogenizer and cell debris werediscarded with a centrifugation step. One milliliter and a half of thissoluble fraction was injected on the NHS-clone 6 immobilized column at aflow rate of 0.5 ml/min. Non specific proteins were washed away with 40ml of running buffer and PulD-N was eluted with an acidic pH jump usinga 100 mM glycine buffer (pH 2.5, 250 mM NaCl). Purity of the elutedprotein was checked by loading fractions on a SDS gel.

Far Western Blotting

Outer membranes of E. coli PAP105+/− PulD (pCHAP3671; (Guilvout et al.,1999)) or PulD-CS (pCHAP3711; (Guilvout et al., 2006)) together withPulS (pCHAP580; (Daefler et al., 1997)) were prepared as before.Membranes were resuspended and stored in 50 mM Tris-HCl, pH 7.5,containing 10% sucrose and 0.1 mg/ml of the protease inhibitor Pefabloc(Interchim, Montluçon, France) and were subjected to SDS/PAGE andtransferred onto nitrocellulose sheets that were then blocked andincubated with periplasmic (osmotic shock) extracts of strains producingSac7-PhoA chimeras. After washing, bound PhoA was detected by antibodiesagainst PhoA, horseradish peroxidase-coupled secondary antibodies, andchemiluminescence.

Secretion

Strain PAP7232 (Hardie et al., 1996) was transformed with the emptyvector or with plasmids encoding Sac7-PhoA chimeras. Transformants weregrown in medium containing 0.4% maltose (to induce production ofpullulanase and its secretion system, including PulD) and 1 mM IPTG toinduce Sac7-PhoA production. Secretion levels were measured as describedin (d'Enfert et al., 1989) and are expressed as the amount of enzymeactivity detected in whole cells compared with that detected in lysedcells (100%). Cell extracts were also examined by immunoblotting withantibodies against PulD and PhoA.

To analyze the effects of the Sac7-PhoA chimeras at higher(plasmid-encoded) levels of PulD production, a zeocin resistance genewas amplified with flanking PstI sites and inserted into the unique PstIsite in the blaM gene of the corresponding plasmids. The recombinantplasmids were then transformed together with pCHAP231 (d'Enfert et al.,1987) into the envelope protease-deficient strain PAP5198 (degP, ompT,ptr). Pullulanase secretion and levels of PulD (with or without priortreatment with phenol) were analyzed as above with or without inductionby IPTG.

Results

Example 1 Design of the First Generation Library (Library 11)

The first critical step for investigating the possibility to use Sac7das a scaffold for obtaining binders to various ligands was to design alibrary by randomization of the potential binding area, whilemaintaining the stability and the solubility of the parent Sac7dprotein.

Several three dimensional structures of Sac7d-DNA complexes are known(Agback et al., 1998; Edmondson and Shriver, 2001; Gao et al., 1998;McAfee et al., 1995; McCrary et al., 1996; Robinson et al., 1998; Su etal., 2000) (FIG. 1 a). According to these structures, the binding ofSac7d to minor groove of DNA involves a binding surface area thatmatches remarkably (shapes and charges) with DNA. This binding area iscomposed of sixteen residues (K7, Y8, K9, K21, K22, W24, V26, K28, M29,S31, T33, K39, T40, R42, A44, S46) (FIG. 1 b). A visual inspection ofthis binding area shows that it is twisted and that it comprises twokinds of geometries: one lightly concave (K7, Y8, K9, W24, V26, K28,M29, S31, T33, R42, A44, S46) and the other essentially flat (K21, K22,W24, T33, K39, T40, R42). Residues W24, T33, and R42 are shared by thesetwo surfaces. About a quarter of the sequence of Sac7d is dedicated tothe binding of DNA. Although all of these residues are exposed on thesurface, a massive random mutagenesis of 25% of the sequence of Sac7dcould be dramatic for the folding and the stability of the mutantsobtained. In a first step, the inventors decided to exclude K28 and K39from the mutagenesis scheme, since these two residues are not fullyorientated towards the binding surface. The second reason to excludethem was that generation of libraries by a one-step PCR as describedbelow would have been impossible due to reduced overlaps available forprimers annealing (see below). It was also postulated that the geometryof the concave side of the binding area could match well with thespherical shape of globular proteins, or, at least, that it couldaccommodate binding of their exposed loops. Thus, the substitutionstrategy focused on the eleven residues in this region of Sac7d (K7, Y8,1K9, W24, V26, M29, S31, T33, R42, A44, S46).

The gene coding for Sac7d is quite short (about 200 base pairs). Thus,the library corresponding to random substitutions of the eleven residuescould be obtained by a one-step PCR. This was done using a mixture ofthree degenerate oligonucleotides (NNS scheme) and three standardoligonucleotides. The codons of the randomized positions were encoded byNNS triplets that allow representation of all amino acids. With thismutagenesis strategy, the theoretical diversity is about 3.2 10¹⁶(32¹¹), which exceeds by about four orders of magnitude theexperimentally achieved diversity of our library. Indeed, according tothe amount of PCR assembly product used to generate the ribosome displayconstruct, the upper estimate of the library was about 3.0 10¹²variants. Sequencing of forty random clones confirmed that the observedresidue frequency was similar to that predicted (data not shown). Thepercentage of correct clones, without any frame shifts or deletions, wasfound to be around 50%. Hence, the “functional” diversity was consideredsatisfactory and the library was used for selections.

Example 2

Ribosome Display Selections using Library 11

Three proteins were chosen as targets: DnaK, GarA (both fromMycobacterium tuberculosis and the N-terminal domain of PulD (fromKlebsiella oxytoca). PulD is an outer membrane protein that cannot bemaintained in solution without ionic detergents. Therefore, a solublemonomeric fragment was used. This fragment, named PulDN, corresponds tothe N-terminal region of PulD. These targets were chosen becausespecific and avid binders could be useful tools to study DnaK, GarA andPulD. Furthermore, these proteins are difficult to crystallize forstructural studies. Binders recognizing these proteins could be used forco-crystallization trials in the same way as antibody fragments(Ostermeier et al., 1995).

ELISA plates coated with neutravidin were used to immobilizebiotinylated proteins and to perform selections. After four rounds ofselection, enrichment with specific binders for all three targets wasobserved (FIG. 6 a). In all cases, more than 50% of binding of pools wasinhibited with 10 μM of free targets, according to RIAs.

To evaluate pools of binders further by ELISA for the selection withPulDN, the RT-PCR products from selected pools were cloned into pQE30vector (Qiagen) using BamHI and HindIII restriction sites. The resultingligations were used to transform E. coli DH5α strain. 96 randomly cloneswere picked from Petri dish to inoculate a deep-well plate containing1.5 ml of LB medium per well (100 μg/ml ampicillin, 1% glucose). Afterovernight culture at 37° C. with shaking at 250 rpm, 0.2 ml of each wellfrom this master plate was used to inoculate an other deep-well platecontaining 1.3 ml 2YT medium (100 μg/ml ampicillin) per well. The platewas then incubated at 37° C. for 1 h with shaking (250 rpm). Theexpression was induced with the addition of 0.5 mM IPTG and incubationat 30° C. for 4 h with shaking (250 rpm). Cells were pelleted with acentrifugation step (2250g) and supernatants discarded. Proteins wereextracted with 50 μl BugBuster (Novagen) per well with shaking at 250rpm for 30 min, then 250 μl of TBS pH 7.4 (20 mM Tris-HC1, 150 mM NaCl)were added. Cell debris were pelleted with a centrifugation step (2500g). For ELISA screening, 100 μl of each supernatant were used to testthe binding on PulD-N or BSA coated into a Maxisorp plate. The detectionwas performed using the RGS His antibody HRP conjugate (Qiagen) thatdetects only the RGS-(His)6-tag (SEQ ID NO: 64) from binders and not the(His)6-tag (SEQ ID NO: 65) from target proteins and BM-Blue substratefrom Roche. All incubation steps were carried-out in TBS pH 5 7.4 with0.1% Tween 20. For all clones the OD at 340 nm was measured for PulD-Nand BSA binding. Ratio of the value obtained for BSA binding on valuefor PulD-N was calculated for each clone. A signal to noise ratiosuperior to 10 was observed for about 92% of clones from round 4 (FIG. 6b). The binding of 6 purified clones from the PulD-N selection (round 4)was tested for DnaK, GarA, PulD-N and BSA by ELISA. As shown in FIG. 6c, the binding was specific for PulD-N. Sequencing of 28 clones fromround 4 revealed a high diversity of sequences; indeed no sequence wasfound more than once, indicating that a higher selection pressure couldhave been used. Furthermore, no wild type residue could be found at anyof all eleven randomized positions (data not shown). The average yieldof expression estimated after IMAC (immobilized metal ion affinitychromatography) purification of nine clones was about 70 mg/liter flaskculture (4 h induction at 30° C.). The binders could be concentrated atleast up to 45 mg/ml. All in all, these observations suggested that thebinding area of Sac7d is highly tolerant to substitution and that theselected variants retained satisfactory biophysical properties. However,a more detailed analysis by RIA showed that the average affinity forbinders in the pool from round 4 was around 100nM and a fifth roundperformed with a mild (2 h) or a more stringent off-rate selection (17h) failed to increase the average affinity of the anti PulD-N pool (FIG.7).

Such low affinity could be limiting for applications where low nanomolardissociation constants are required. Hence, the inventors decided toexplore other approaches to obtain higher affinity.

Example 3 Design of the Second Generation Libraries (Library 13 and 14)

An intuitive way to improve affinity is to extend the area of bindingand thus to increase the potential number of interactions betweenbinders and their ligands. The potential binding area corresponding torandomization of 11 residues had a 890 A² solvent-accessible surface.Sac7d being very tolerant to 11 substitutions, it was decided torandomize two or three additional positions: K21 and K22 (library 13) orK21, K22 and T40 (library 14) that correspond to 1130 A² and 1200 A²,respectively. Another explanation could be that the concave binding areais not well-suited to bind protein and, therefore, the use of a flattersurface could improve final affinities. These two new libraries (each of3.0 10¹² variants) were constructed the same way as library eleven.Sequencing of seventy random clones from library 14 confirmed that theobserved residue frequency was similar to that predicted, with a slightunder representation of S, L, and R amino acid residues, while P, N, Q,and H amino acid residues were a bit over represented (data not shown).The percentage of correct clones, without any frame shifts or deletions,was found to be around 65%. Hence, the “functional” diversity wasconsidered satisfactory and the library was used for selections.

Example 4

Ribosome Display Selections with PulD-N as Target Protein, using Library13 and 14

Four rounds of selections were performed using these libraries andPulD-N as a target protein. A fifth round was performed in parallel withlibraries 11, 13, 14 with a mild off-rate selection for two hours inorder to enrich the pools for binders with slower off-rates. A radioimmunoassay for the pools after the fifth round (FIG. 7) indicated anenrichment for specific PulD-N binders with the two new libraries.Binding could not be detected on immobilized BSA or neutravidin.Furthermore, this RIA showed that 10 nM of free PulD-N was sufficient toinhibit about 50% of the binding signal with library 13 compared to 100nM PulD-N for the same level of inhibition with library 11. Library 14behaved even better, as 1 nM competitor was sufficient to achievesignificant inhibition of about 20% of the binding signal. Hence, atleast one order of magnitude was gained for the average affinity withthe design used for library 14.

Example 5

Characterization of Anti-PulD-N Binders from Library 14

5.1. Sequence Analysis, Expression And Purification Of Selected Binders

Since the most promising affinities could be found in the pool obtainedfrom library 14 (round 5), the pool of binders from this library wasanalyzed. The enriched pool was cloned into the expression plasmidpQE30, and E. coli strain DH5α was used for protein production.Forty-eight individual clones were assayed by an ELISA procedure usingimmobilized PulD-N or BSA and E. coli crude extracts. For all clones, asignificant and specific binding over background was detected. Thus, allbinders were sequenced for further analysis.

As observed for library 11, a large diversity of binders remained evenafter the fifth round of selection (FIG. 8). However, most of the clonescould be sorted into six families according to their homologies (datanot shown). In addition, two identical clones were found in two cases,all other clones being unique, suggesting a selection convergence. Theresidues found at randomized positions were of very different nature,with aliphatic and aromatic side-chains as well as charged orhydrophilic side-chains. A clear preference for a particular residuecould be observed at some positions. For example, position R42 wasoccupied by a tyrosine in about half of the clones, probably reflectingits importance for the binding of PulD-N. Among all programmedmutations, no strict conservation of the native residue could beobserved, including the three new positions targeted (K21, K22 and T40)compared to library 11. Finally, only seven native residues wereretained out of 560 residues in the forty clones sequenced (40×14).Hence, these observations suggest that the 14 positions targeted alltolerate random substitutions.

The binders accumulated in large amounts in the E. coli cytoplasm at 30°C. after overnight growth and could be purified to homogeneity in onesingle step IMAC with yields up to 200 mg from a one liter shake flaskculture (FIG. 9). The proteins ran on a 15% acrylamide SDS-PAGE gel atthe position expected for their calculated molecular masses. Purifiedbinders could be concentrated up to 60 mg/ml in a standard TBS bufferwith no sign of precipitation and remained soluble over several monthsat 4° C.

Size-exclusion chromatography showed that clones 6, 39, 40 and 41binders studied were monomeric. Like Sac7d, all binders appeared largerthan predicted in this assay (11.5 kDa determined instead of 9.1 kDacalculated), probably due to a deviation from a truly globular shapecaused by the presence of a nine residue N-terminal tag.

5.2. Affinities of PulD-N Binders

In order to identify clones with the highest affinities, forty eightclones were used for micro expressions and IMAC purifications. Thesepurified proteins were then screened by SPR using immobilizedbiotinylated PulD-N on a streptavidin coated chip. For all assayedbinders, no significant binding was observed on the blank surface coatedonly with steptavidin, supporting the idea that the binding was specificfor PulD-N. According to analysis of the dissociation phases, fiveclones with the slowest off-rates were chosen for detailed affinitydetermination of the monovalent proteins by SPR.

These five clones, 6, 33, 39, 40 and 41, were further purified by gelfiltration. Kinetic analyses were performed at different concentrationsand analyzed with a global kinetic fit. The dissociation constants ofall PulD-N binders were found to be in the picomolar or low nanomolarrange (FIG. 10 a and table 3). Clones 6 and 33 had the highestaffinities (K_(D)=130 pM and 190 pM, respectively). The associationkinetics were slightly reduced (factor 1.3) under high ionic strengthconditions (300 mM NaCl), indicating that these associations were notelectrostatically assisted (data not shown). K_(D) values of these fivebinders were confirmed (FIG. 10 b and table 3) by competition SPRanalysis (Nieba et al., 1996). A control experiment showed that nobinding occurred when testing wild type Sac7d for binding to immobilizedPulD-N, indicating that the binding property observed for the selectedclones was the result of a newly introduced function and not the resultof a pre-existing affinity of Sac7d for PulD-N.

TABLE 3 Summary of dissociation constant determinations by surfaceplasmon resonance. Clone k_(on) [M⁻¹ s⁻¹] k_(off)[s⁻¹] K_(D) [nM]^(a)K_(D) [nM]^(b) % acitivity^(c) 6 1.9 10⁷ 2.5 10⁻³ 0.13 0.14 ± 0.0390-100 33 1.7 10⁷ 3.2 10⁻³ 0.19 0.17 ± 0.03 90-100 39 1.7 10⁷ 8.6 10⁻³0.52 1.1 ± 0.2 90-100 40 2.3 10⁷ 1.4 10⁻² 0.60 0.9 ± 0.2 90-100 41 2.010⁷ 1.2 10⁻² 0.61 1.1 ± 0.1 90-100 ^(a)K_(D) values obtained fromkinetic analysis. ^(b)K_(D) values obtained from competition SPRexperiments. ^(c)from competition SPR experiment analysis.

The sequences analysis of these binders revealed different situations(FIG. 8). The randomized positions in clone 6 and 33 were different,despite the fact that their respective K_(D) values were in the samerange (130-140 pM). In contrast, clone 40 had 11/14 randomized residuesin common with those of the clone 41 and had similar K_(D) values (≈1nM). Clone 39 shared 10/14 randomized residues with clone 6 but had a7-fold lower affinity. Interestingly, clone 39 was found to be truncatedby 4 residues at its C-terminal end but remained able to bind PulD-N.This is not surprising, as several truncated forms of Sac7d also exist(McAfee et al., 1995).

5.3. Stability of PulD-N Binders

Sac7d is a hyperthemostable protein that unfolds with a T_(m) of 91° C.at pH 7.0, the thermal stability of the protein decreasing at lower pHs(McCrary et al., 1996). The thermal stabilities of clones 6, 33 and 40were compared to that of wild type Sac7d by differential scanningcalorimetry. In order to insure complete unfolding of the proteins belowthe high temperature limit of the DSC (125° C.), a pH of 5.6 was usedfor all scans. The clones were found thermostable with denaturationtemperatures between 68° C. and 83° C. (FIG. 11 and table 4). Clone 40was remarkably stable with a T_(m) value lower than that of wild-type(89.7° C.) by only 5.8° C., followed by clone 33 ΔT_(m)=−10.5° C.), theleast stable clone being clone 6 (ΔT_(m)=−22.0° C.). However, T_(m) ofclone 6 still remains 8° C. above the mean T_(m) value of proteins ofthe Protein Data Bank (Freire, 2001). DSC scans were characteristic ofcooperative unfolding indicating that, although with a high mutationload (up to 14 residues mutated, i.e. 21% of Sac7d mutated), Sac7dvariants were able to adopt a folded structure as for the wild-type. Thecalorimetric enthalpy (the heat change per mole, ΔH_(cal)) was lowerwith respect to wild-type (42.5 kcal.mol⁻¹) by only 5.8 and 4.8kcal.mol⁻¹ for clone 40 and clone 33, respectively (table 4).Furthermore, for both clone 40 and clone 33, β, the ratio value of thevan't Hoff enthalpy (the heat change per cooperative unfolding unit) tothe calorimetric enthalpy was close to one, and close to the β value ofwild-type Sac7d. This observation strongly indicated that unfolding ofclone 40 and clone 33 corresponded to that of the folded protein monomer(table 4). Clone 6 presented a lower calorimetric enthalpy with respectto wild type (by 18.10 kcal.mol⁻¹) and a higher β ratio (table 4). Theseobservations may be related to the partial unfolding of clone 6 proteinat acidic pHs where the protein is less stable (T_(m) of clone 6 is 8°C. higher at pH 7; data not shown). Taken together, these observationsshow that clones 6, 33 and 40 retained to a large extent the favorablethermal stability of the wild type protein.

TABLE 4 Thermal unfolding parameters for wt and variants Sac7d CloneT_(m) [° C.]^(a) ΔH_(cal) [kcal · mol⁻¹]^(b) ΔH_(vH) [kcal · mol⁻¹]^(b)β^(c) 6 67.7 24.4 46.3 1.9 33 79.2 37.7 45.2 1.2 40 83.9 36.7 47.7 1.3Sac7d wt 89.7 42.5 55.2 1.3 ^(a)T_(m) is ±0.1° C.; ^(b)ΔH_(cal) is ±0.3kcal · mol⁻¹, ΔH_(vH) is ±0.5 kcal · mol⁻¹; ^(c)β = ΔH_(vH)/ΔH_(cal)

5.4. Specificity of PulD-N Binders

How specific are these PulD-N binders, and can they be used forbiotechnological applications in which specificity is crucial?

To answer these questions, binders 6, 33 and 40 were fused to theN-terminus of E. coli alkaline phosphatase (PhoA). The chimeras(Sac7*-PhoA) were produced as periplasmic proteins in strain DH5α usingpQUANTagen vector encoding the PhoA signal peptide. The chimeras werethen extracted from the periplasm by osmotic shock and theirfunctionality assayed by ELISA with PulD-N- or BSA-coated wells. Boundfusions were quantified with chromogenic p-nitrophenylphosphatesubstrate and spectrophotometry (data not shown). A high signal overbackground was observed for all three chimeras (ratiosignal/background>10). This showed that the chimeras were 1) exported inthe periplasm, 2) still able to recognize specifically PulD-N and 3)catalytically active. Hence, these fusions could be used as a singlestep ELISA detection reagent.

To evaluate if these fusions are able to discriminate the PulD-N proteinin a complex mixture of proteins, such as in an E. coli crude extract,we used them as a detection reagent for immunoblots. In this experiment,an overnight culture of plasmid-free DH5α was harvested and lysed. Thecrude extract was aliquoted and decreasing amounts of purified PulD-Nwere added to each tube. After SDS-PAGE and transfer to a nitrocellulosemembrane, PulD-N was detected with the binder-PhoA chimeras in presenceof the precipitating chromogenic substrate (NBT/BCIP) in amounts as lowas 1.5-3 ng (FIG. 12 a) without crossreactivity. This supported thedouble functionality of fusions observed with ELISA (FIG. 12 a). Thedetection of low amounts of PulD-N was done while far higher amounts ofendogenous proteins were present, demonstrating that the binders 6, 33,and 40 were able to discriminate PulD-N among thousands of proteins andwere therefore highly specific.

To evaluate specificity further, binder 6 was covalently immobilized viaan amine coupling reaction on a one milliliter NHS-agarose activatedcolumn. The soluble fraction of an E. coli crude extract correspondingto 40 ml of a culture producing PulD-N was then injected onto thecolumn. Fractions were collected during loading, washing and elution(acid pH jump) steps. The SDS-PAGE analysis of these fractions showedthat the column was able to trap the PulD-N present in the crude extractand that this was done with high specificity, since the only visibleband on the stained SDS-PAGE gel corresponded to PulD-N (FIG. 12 b).Thus, the ability of the binders to discriminate PulD-N from thousandsof proteins was again confirmed.

Example 6 Binding of PulD-N Binders to Dodecameric PulD

Next, the inventors investigated if the three selected Sac7d derivativeswith the highest affinities for PulD-N (binders 6, 33 and 40) were ableto recognize the full-length PulD protein integrated in to the E. coliouter membrane. Full-length PulD forms dodecamers in the membrane (whilePulD-N is monomeric) (Chami et al., 2005) and it could not be excludedthat the epitope recognized by each of those binders affected bymutlimerization of PulD, thus preventing their binding to native PulD.When increasing amounts of PulD-containing membranes from E. coli PAP105(pCHAP3671 pCHAP580) were mixed with saturating amounts of GFP-taggedbinders 40 or 33, the amount of binder remaining in the pellets aftercentrifugation was correspondingly increased (FIG. 13A). The GFP-taggedbinders were not sedimented with membranes from PAP105 (pCHAP3711pCHAP580) containing a PulD variant lacking the N-domain (Guilvout etal., 2006) (FIG. 13A). Thus, binding of these GFP-binder chimeras tomembranes is PulD-N-specific, and they bind to the native, dodecamericsecretin complex despite the presence of the GFP tag. The binder 6 boundonly very weakly to membranes containing PulD (data not shown).

A far-Western immunoblot was used to validate binding of the Sac7dderivatives to PulD dodecamers. All three Sac7*-PhoA chimeras boundspecifically to both monomeric and dodecameric PulD but not to PulD-CS(FIG. 13B). Although these three chimeras bound equally well tophenol-dissociated (monomeric; see (Hardie et al., 1996)) PulD (data notshown), they consistently exhibited different apparent affinities fordodecameric PulD, ranging from high (binder 40), to low (binder 6).

Example 7 PulD-N Binders Inhibit Pullulanase Secretion and Prevent PulDMultimerization

Sac7*-PhoA chimeras, in which Sac7d or its derivatives are sandwichedbetween the PhoA signal peptide and the catalytic part of PhoA, wereefficiently exported to the periplasm, as monitored by the high PhoAactivity and release upon periplasmic shock. Plasmids encoding theSac7*-PhoA chimeras were transformed into E. coli strain PAP7232, inwhich the pul genes are integrated into the chromosome. All threechimeras were produced in similar amounts after IPTG induction andinhibited pullulanase secretion completely, whereas exported Sac7d-PhoAwas without effect. Furthermore, neither PulD dodecamers nor monomerswere detected in strains producing any of the Sac7*-PhoA chimeras (datanot shown). To obtain more-precise information on these phenomena and tostudy the fate of PulD in strains producing the chimeras, they wereproduced in envelope protease-deficient strain PAP5198 carrying pCHAP231(to increase T2SS production; see Guilvout el al., 2006). IPTG-inducedlevels of Sac7*-PhoA production inhibited pullulanase secretion by >90%(FIG. 14A). Dodecameric PulD was much less abundant, and PulD monomerswere correspondingly more abundant (arrows in FIG. 14B) than in controlswithout chimeras or with Sac7d-PhoA, demonstrating that the chimerasprevent PulD multimerization and cause PulD monomer degradation byenvelope proteases. Similar results were obtained with uninduced levelsof Sac7*33-PhoA and Sac7*40-PhoA, but substantial pullulanase secretion(>50%) and PulD multimerization occurred when Sac7*6-PhoA was present atuninduced levels (FIG. 14C).

Example 8 Selection of Sac7d Variants Binding to Other Targets

The best library (library 14) was used to obtain binders specific forother targets, by using the same technology as described above.

Selections were carried out by anti-protein ribosome display focusing onthe following proteins: PulDN1 (26.8 kDa), NGF (13 kDa), PknG (81,6kDa), GarA (12 kDa) and lysozyme (14.3 kDa) up to cycle No. 5. Duringthe 4^(th) cycle, the selection process was conducted in parallel, withand without the target. FIG. 15 shows that, after 4 selection cycles, aPCR product is obtained only when the target is present (lanes marked as“+”). This suggests that the selection pools were enriched with clonesspecific for each of the targets tested.

In order to further evaluate the success of the selection process, acompetitive radio immuno assay (RIA) was carried out using the targetproteins at various inhibitory concentrations. These RIAs clearly showthat there is marked enrichment for anti-PknG, anti-lysozyme andanti-PulDN1 selections whereas in the case of NGF and GarA, the resultssuggest that the selection has not yet sufficiently converged. FIG. 16shows that the average anticipated affinity for the anti-PknG andanti-lysozyme variants is of the order of 1 to 10 nM.

The anti-lysozyme, anti-PknG and anti-GarA selection pools used in cycleNo. 5 were screened according to the ELISA method by preparing 96-wellmicro-cultures and using bacterial lysis supernatants after inducingclone expression with IPTG for 4 hours at 30° C., The results presentedin FIG. 17 clearly show that there are specific clones for each of thetargets. Although the RIA for GarA was not encouraging, a significantproportion of positive clones were nevertheless detected with the ELISAmethod. A particular clone binding GarA was isolated and sequenced. FIG.19 shows the alignment of this binder's sequence(GSVKVKFLYLGEEKEVDTSKIWFVMRAGKHVYFQY DDNGKYGIGWVREKDAPKELLDMLARAEREKKL,SEQ ID No: 47) with Sac7d.

As regards PknG, a significant proportion of clones appear to bind toBSA, which suggests that an additional selection cycle is required inorder to eliminate them. Anti-PulDN1 and anti-NGF screenings are carriedout.

The positive anti-lysozyme clones were screened and classed by Biacorefor the best anticipated affinities (long complex dissociation time(k_(off))), by preparing 96-well microcultures. The 24 best clonesselected were then sequenced (FIG. 18). A preferential sequence, such asthat of the Lys_B11 clone, is clearly evident since it is representedseveral times (although encoded for certain positions by differentcodons for the same amino acid). The screening of cycle No. 4 revealed agreater diversity (not shown).

Three clones of different sequences (Lys_B3, Lys_H4 and Lys_H8) wereproduced in E. coli, purified on a large scale by IMAC and then passedthrough a molecular sieve. The production levels obtained wereapproximately 120, 40 and 25 mg/L of culture, respectively. Theaffinities determined by Biacore are being obtained and processed.Current (preliminary) data indicate affinities of the order of 15 nM, 3nM and 25 nM (Lys_B3, Lys_H4 and Lys_H8, respectively).

The three anti-lysozyme clones (Lys_B3, Lys_H4 and Lys_H8) are currentlybeing characterised by microcalorimetry in order to determine theirthermostabilities and affinities in solution.

The clones obtained from other selections will also be screened byBiacore and sequenced, and the affinities of certain clones will bedetermined more precisely by Biacore.

Example 9 Selection of Sac7d Variants Binding to a Human IgG Fc Fragment

9.1. Selection of Fc Binders

A Human IgG Fc fragment (MW=50 kDa) provided by Bio-Rad was chemicallybiotinylated using sulfosuccinimidyl-6-(biotinamido) hexanoate. Thedegree of biotinylation, determined by a HABA assay, was about 2 to 3molecules of biotin per protein molecule. Biotinylated Fc was bound toimmobilized neutravidin or streptavidin in a Maxisorp plate (Nunc) andselections by ribosome display were performed at 4° C. using the libraryderived from Sac7d (as described above). Four rounds of selection wereperformed to isolate binders. Neutravidin and streptavidin were usedalternatively from round to round to avoid unspecific binders.

9.2. Evaluation of the Pool of Sequences Selected

After a round of selection, a pool of sequences (supposedly binders)carrying an RGS-His6-tag (SEQ ID NO: 64) was obtained. This pool wastranslated in vitro using an E. coli S30 extract, and its bindingactivity was tested by ELISA using an anti-RGS-His6-tag-HRP (SEQ ID NO:64) conjugate. A binding activity was detected in the pool of selectionagainst passively immobilized Fc fragment, and no binding signal wasobserved against BSA, Neutravidin and Streptavidin (not shown). Thisresult suggests that the pool of binders is probably specific of Fefragment and that there is no significant contribution of Neutravidin orStreptavidin to binding activity.

A competition ELISA with this translated pool was also carried-out toroughly evaluate the average affinity of selected binders. In thisexperiment, the translation was pre-incubated with severalconcentrations of Fc fragment before incubation in the ELISA plate inwhich Fc fragment was immobilized. As shown in FIGS. 20, 20% and 70% ofthe signal could be inhibited with 10 nM and 100 nM Fc, respectively.This result suggests that expected average affinity is around dozens ofnM. This range of affinity is similar to what was observed for PulD-Nselection.

Example 10 Automation of the Technology

To increase the potential of the above-described technology, theribosome display selection protocol (FIG. 21) is transferred to anautomated platform for the subsequent, simultaneous and rapid testing ofnumerous targets (still using library 14, or any other library, forexample starting from an OB-fold protein different from Sac7d).

A Tecan Gemini 150 robot has been fully equipped to this aim. Thisstation comprises: several heating/cooling/agitating blocks, a MJresearch thermocycler for automated station with motorized lid, all theneeded accessories to perform reaction set-ups, RNA and DNA cleanups aswell as to measure concentrations of nucleic acids with an integratedmicroplate reader. The goal is to achieve one round of selection withoutmanual intervention at all. Manual intervention would only be requiredbetween the rounds of selection as many plastic ware have to be replacedat the end of a cycle. Manual selections by ribosome display (FIG. 21)are repetitive (five rounds necessary to enrich for binders), fastidious(many steps of transcription, translation, PCR, RT-PCR) and thereforetime consuming for few targets, or even not manageable for more thanfour targets by one experimentalist. Hence, there is a great practicaladvantage to perform selections using a robot. After validation of theselection protocol on the robotic station (with PulD target), it ispossible to select binders against up to 48 targets on a time scale ofone or two weeks.

Discussion

The inventors explored potential benefits of what evolution hasaccomplished with OB-folds during millions of years to adapt this foldto bind a wide diversity of ligands: metallic ions, sugars, nucleicacids (RNA, single and double stranded DNA) and proteins. They were ableto show that a member of the OB-fold family can indeed be converted fromDNA recognition to protein recognition by in vitro evolution. This ledsuccessfully to the generation of high affinity, high-specificitybinders for a given target.

Design of Sac7d Libraries

The observation that the OB-fold architecture is polarized, meaning thatthe binding face is always the same in all OB-fold proteins (Arcus,2002), enabled the design of libraries of Sac7d variants retaining thefavorable biophysical properties of the wild type protein. DifferentOB-fold proteins can exhibit substantial variation in the lengths ofloops 1, 3 and 4 (FIG. 1 b and 2), which are often implicated in ligandbinding. Although most scaffolds used up to now are based onrandomization of flexible loops similarly to those in antibodies (Binzet al., 2005), the inventors decided to keep the original loop lengthsfrom the native Sac7d protein. They first postulated that the lightlyconcave part of the binding area would be best adapted to globular shapeof proteins used as targets. However, as reported above, therandomization of the residues from this area did not allow to obtainbetter than high nanomolar binders. This potential binding surfacecorresponds to a solvent-accessible surface area to of about 890 A² andis in the typical range for antibody-protein associations (777±135 A²)(Jones and Thornton, 1996). Thus, this surface should be sufficient toprovide enough energy of interaction to get monomeric binders with highaffinity. The extension of the potential binding area with three moresubstitutions was then tested.

Selection with Sac7d Libraries

Selections of binders against several different targets weresuccessfully carried out using ribosome display. In contrast toselections with fully synthetic naive scFv libraries, for which 5 to 6rounds were necessary for enrichment (Hanes et al., 2000), a significantenrichment was observed already after the third round with Sac7dlibraries. This could be explained by a more efficient folding of Sac7dcompared to antibodies, as it requires neither disulfide bond formationto reach its native state nor the correct association of two linkedstructural domains (as for scFv fragments) to be functional. The highrate of enrichment is similar to that described for the DARPin scaffold(Binz et al., 2004a). Finally, outputs of selections against PulD-Ndemonstrate that the libraries contain sufficient functional diversityto provide different binders able to recognize a same target protein.This validated the design of Sac7d libraries according to the invention.

Properties of Selected Binders

Characterization of the selected monomeric binders against PulD-N showedthat picomolar affinities can be obtained with Sac7d libraries. Theseaffinities are among the highest obtained with a scaffold proteinwithout the need for an affinity maturation step (for a review, see(Hosse et al., 2006) and references therein). The kinetics of bindingswere also remarkable, with rates of association around 2 10⁷ M⁻¹.s⁻¹that rank PulD-N binders in the upper range of diffusion-limitedassociation rates for protein-protein interactions (Gabdoulline andWade, 1997). The rates of association do not appear to beelectrostatically assisted. Thus, the high rates of association could beexplained by the “preformed” shape of the binder that matchesgeometrically to the target without requiring conformational changes.

The high affinities obtained are associated with a very highspecificity. The inventors have shown that whatever the context (E. colicrude extract or membranes) and the approach (immunoblot or affinitychromatography), all tested binders were able to discriminate the targetamong thousands of other proteins. This stringent specificity could berelated to the rigidity of the scaffold that prevents slight adaptationsto different targets. Furthermore, all of the dozens of binders screenedwere shown to be target-specific in ELISA assays, indicating that theselection pressure applied was sufficient to get rid of stickymolecules.

Recombinant protein yields for variants (up to 200 mg/l E. coli culture)were much higher than reported for Sac7d (about 10-15 mg/l). Thisdifference can be explained by the lysis that occurs few hours afterinduction of the expression of the wild-type sac7d gene. This toxicityis probably due to perturbations of regulation pathways induced by Sac7dbinding to the E. coli genome, as Sac7d is a general DNA bindingprotein. This limitation is not seen for variants, suggesting thatvariants had indeed lost their DNA binding property.

The thermodynamic stability observed for three variants compares wellwith several other proteins from thermophiles (Kahsai et al., 2005) andthe thermal stability of the binders remained close to that of wild-typeSac7d. Clone 6, the least stable clone analyzed, still remained athermostable protein with a T_(m) of about 68° C. at pH 5.6, a T_(m) by8° C. lower compared to that at pH 7.0. Hence, it appears that despitethe variable T_(m) values observed from clone to clone, globally thelibrary design described above, combined with the use of a protein of anextremophile origin, led to the generation of very stable proteins withthe desired recognition properties.

Binding In Vivo and Intracellular Inhibition of Pullulanase Secretion

All three Sac7*-PhoA chimeras that were tested bound to monomericfull-length PulD. Sac7*40-PhoA and Sac7*33-PhoA bound well tododecameric PulD, indicating that their epitopes remain accessible. Incontrast, Sac7*6-PhoA bound only weakly to PulD dodecamers but had thehighest affinity for PulD-N in vitro, indicating that its epitope ispartially masked upon multimerization and is different from thoserecognized by Sac7*40 and Sac7*33. ITC competition experiments indicatedthat epitopes recognized by Sac7*6 and Sac7*40 are identical or overlap(data not shown). The differences in the in vivo effects of these twobinders, when fused to PhoA, suggest that their epitopes overlap.

All three Sac7*-PhoA variants prevented PulD multimerization andtargeted the PulD monomers for degradation by envelope proteases,thereby blocking pullulanase secretion. Earlier evidence indicated thatthe N domain does not influence PulD multimerization (Guilvout et al.,2006). PhoA dimerization in Sac7*-PhoA might cause steric hindrance andconsequent mispositioning of PulD monomers. Secretion levels in strainsproducing Sac7*-PhoA remained very low when the level of PulD producedwas increased and envelope proteases were inactivated (FIG. 14B). Lowsecretion could be due to the presence of only a few PulD dodecamers,channel occlusion, or masking of an essential interaction site withsubstrate (Shevchik et al., 1997) or another secreton component (Possotet al., 2000) by bound chimeras.

Reducing the level of Sac7*33 or Sac7*40 (by eliminating induction byIPTG) did not diminish their effect on secretion or PulDmultimerization, but Sac7*6-PhoA was almost without effect under theseconditions (FIG. 14C), even though it was at least as abundant as theother chimeras (FIG. 14A). Two different scenarios could explain thisobservation. First, the highly abundant Sac7*6-PhoA binds to almost allPulD monomers and prevents their multimerization. However, whenSac7*6-PhoA levels are lower, it cannot compete efficiently with PulDmonomer-monomer interactions, and enough multimers assemble to allowefficient secretion. The apparently lower affinity of Sac7*6-PhoA fordodecameric PulD is insufficient to prevent secretion. Second, bindingof the chaperone PulS to PulD monomers, a prerequisite for their correcttargeting to the outer membrane (Guilvout et al., 2006; Hardie et al.,1996), prevents Sac7*6-PhoA binding and permits correct multimerization.In this scenario, the outcome depends on which protein binds first toPulD, PulS or Sac7*6-PhoA. Both scenarios are in agreement with the factthat the epitope recognized by Sac7*6 is strongly masked upon PulDmultimerization, suggesting that it is at the interface between twomonomers.

Potential Biotechnological Applications

These binders retained most of the very favorable biophysical propertiesrequired in an alternative scaffold to antibodies. Their propertiescompare well with previously proposed alternatives to antibodies such asaffibodies (Nord et al., 1997), fibronectin (Xu et al., 2002), orankyrins (Binz et al., 2004a). Indeed, they are very well expressed inE. coli (in the cytoplasm or the periplasm), stable, soluble, able torecognize a protein target with a high affinity and a high specificity,and they can be fused functionally to different reporter proteins (PhoAand GFP). In other words, they are cheap to produce, easy to purify andto handle. This opens the door for a number of biotechnologicalapplications. Furthermore their very small size, three or nineteen timessmaller than a scFv or an IgG, respectively, makes them ideal candidatesfor the design of chimeric proteins in which binding modules are neededwhile maintaining a reasonable size. It is also conceivable to linkseveral binders with different specificities and, thus, to constructfusions with multiple specificities. Another advantage of small binderssuch as Sac7d is their potential ability to bind buried epitopes thatare sterically inaccessible to natural antibodies or their fragments.

It can be noted that the capacity of the binders to bind to targetproteins is not limited to targets with a high molecular weight. Indeed,the results already obtained, in addition to those concerning PulD, showthat the bank developed from Sac7d has allowed the isolation of clonesspecifically binding to three new targets, which cover a wide range ofmolecular weights (Lysozyme =14.3kDa, GarA =17.3kDa, PknG =81.6kDa). Theexpression of these binders in E. coli is far superior to the levelsobserved for recombinant antibody fragments. The affinities obtained areof the order of nM without selection based on long k_(off) values(off-rate selection), due to a shortage of time. This selection step isneeded in order to obtain sub-nanomolar affinities, and is currentlyperformed.

To address how specific could be PulD binders, the inventors havealready demonstrated they could be used for the development of detectionreagents. For example, one step ELISA or Western blots are achievable byfusion of binders to alkaline phosphatase. Another example is the use ofGFP fusions for in vitro detection that paves the way to in vivointracellular localization. It was also demonstrated that affinitychromatography can be performed with these binders and that a proteincan be purified to homogeneity by single step purification. Otherapplications could use these binders, such as protein chip arrays,biosensors or switchables in vivo knock out for example.

Conclusions

The strategy to use an OB-fold protein to get binders able to recognizetarget proteins with high affinity and very fine specificity wassuccessfully validated by the above results. The starting point wasSac7d, a general DNA binding protein. Sac7d and its derivates are nowable to recognize two structurally unrelated families of ligands: DNAand proteins. Hence, the inventors have reproduced in vitro the bindingadaptation of an OB-fold for a given target, such as Nature as alreadyachieved. It is therefore conceivable, that Sac7d might also be adaptedto other known ligands of OB-fold proteins, such as single strand DNA,RNA or sugars. In addition to their potential use in functional knockoutexperiments, these binders could be used for affinity chromatography,detection, in vivo localization experiments, etc. Indeed, the favorablebiophysical properties of these binders, along with their facile fusionto different reporter proteins and their small size (3 and 19 timessmaller than scFv and IgG, respectively), might facilitate theseapplications.

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1-27. (canceled)
 28. A method for obtaining a molecule binding to atarget of interest, comprising the steps of a. providing a combinatoriallibrary of variants of a starting OB-fold protein in which 5 to 32residues involved in the binding of said starting OB-fold protein withits native ligand have been randomized, wherein said starting OB-foldprotein is selected from the group consisting Sac7d or Sac7e fromSulfolobus acidocaldarius, Sso7d, from Sulfolobus solfataricus, DBP 7from Sulfolobus tokodaii, Ssh7b from Sulfolobus shibatae, Ssh7a fromSulfolobus shibatae, and p7ss from Sulfolobus solfataricus, and whereinsaid residues that are randomized in said variants are selected amongstthe residues corresponding to V2, K3, K5, K7, Y8, K9, G10, E14, T17,K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40,R42, A44, S46, E47, K48, D49, A50 and P51 of Sac7d; b. exposing saidvariants to said target of interest; c. selecting a variant of saidstarting OB-fold protein which binds to said target of interest therebyobtaining a molecule binding to said target of interest.
 29. The methodof claim 28, wherein said selection step is performed by a methodselected from the group consisting of phage display, mRNA display,bacterial display, yeast display and ribosome display.
 30. The method ofclaim 28, wherein said target of interest is selected from the groupconsisting of a peptide, a protein, an oligosaccharide, asingle-stranded DNA, a double-stranded DNA, an RNA, a metallic ion, anda carbohydrate.
 31. The method of claim 28, wherein said variantsfurther comprise an insertion of 1 to 15 random amino acid residues inloop 3 and/or an insertion of 1 to 15 random amino acid residues in loop4, and/or an insertion of 1 to 20 random amino acid residues in loop 1.32. The method of claim 28, wherein 11, 12, 13, 14, 15, 16, 17 or 18residues are randomized, chosen amongst residues corresponding to K7,Y8, K9, K21, K22, W24, V26, K28, M29, S31, T33, K39, T40, R42, A44, S46,E47 and K48 of Sac7d.
 33. The method of claim 28, wherein 11, 12, 13,14, 15 or 16 residues are randomized, chosen amongst residuescorresponding to K7, Y8, K9, K21, K22, W24, V26, K28, M29, S31, T33,K39, T40, R42, A44 and S46 of Sac7d.
 34. The method of claim 28, inwhich the randomized residues in said variants comprise at least theresidues corresponding to K7, Y8, K9, W24, V26, M29, S31, T33, R42, A44and S46 of Sac7d.
 35. The method of claim 28, wherein only residuescorresponding to K7, Y8, 1(9, W24, V26, M29, S31, T33, R42, A44 and S46of Sac7d are randomized in said variants.
 36. The method of claim 35, inwhich one, two or three additional residues selected amongst residuescorresponding to K21, 1K22 and T40 of Sac7d are also randomized in saidvariants.
 37. The method of claim 28, wherein residues corresponding toK7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, R42, A44 and S46 of Sac7dare randomized in said variants.
 38. The method of claim 28, wherein 1to 15 random amino acid residues are inserted in the regioncorresponding to the amino acids in position 25 to 30 of Sac7d, and/or 1to 15 random amino acid residues are inserted in the regioncorresponding to the amino acids in position 35 to 40 of Sac7d, and/or 1to 20 random amino acid residues are inserted in the regioncorresponding to the amino acids in position 7 to 12 of Sac7d in saidvariants.
 39. The method of claim 38, wherein 1 to 15 random amino acidresidues are inserted between the amino acids corresponding to aminoacids G27 and K28 of Sac7d, and/or 1 to 15 random amino acid residuesare inserted between the amino acids corresponding to amino acids N37and G38 of Sac7d, and/or 1 to 20 random amino acid residues are insertedbetween the amino acids corresponding to amino acids K9 and G10 of Sac7din said variants.
 40. The method of claim 33, wherein 1 to 4 amino acidresidues selected amongst residues corresponding to A59, R60, A61 andE64 of Sac7d are deleted in said variants.